This proposal is to develop methods and instrumentation for the simultaneous imaging and optical stimulation of activity in large populations of neurons in the awake mouse brain under conditions in which the mouse is free to navigate in a virtual environment, a capability with wide application in systems neuroscience. The enabling technology is an apparatus that facilitates high-resolution optical imaging at cellular resolution while minimizing brain motion. It is based on an upright table mounted two-photon microscope mounted over a spherical treadmill consisting of a large air supported ball. Mice, with implanted cranial windows designed to reduce brain motion, can walk and run on the surface of the ball during imaging while their head remains motionless. Image sequences demonstrate that movement-associated brain motion is limited to ~2-5 um and that this motion is predominantly in the focal plane, with little out-of-plane motion, providing the conditions for an offline Hidden Markov Model based software method for removing residual motion artifacts. Pilot data using a first generation instrument demonstrate that behaviorally correlated calcium transients from large neuronal and astrocytic populations can be routinely imaged at cellular resolution in awake mice, even during walking and running. The proposed research and development program will further validate and optimize the methods used in the first generation instrument, and extend its capabilities by adding real-time motion correction, new chambers and lenses for imaging of deeper brain structures such as the hippocampus, and the incorporation of a visual virtual reality display system controlled by the running behavior of the mouse. An additional thrust will focus on using pulse time-multiplexing and a multi-focal plane optical design to provide simultaneous imaging and two-photon based photo-stimulation using channelrhodopsin. The scientific questions in systems neuroscience that can be addressed using the instrumentation to be developed are some of the most fundamental ones about how the brain works, ranging from determining the number of active, versus silent, neurons during a specific behavior, to the importance of synchrony and correlation in perception, memory, and motor control. The ability to image the activity in entire populations of neurons at cellular resolution in mice navigating in a virtual environment will facilitate mapping the micron-scale spatial architecture and circuit connectivity of place cells in the hippocampus and grid cells in the cortex. This study will develop an imaging method to measure the chemical processes in many individual neurons simultaneously in the brain while it is operating in the awake state. This capability would be valuable in comparing normal and diseased states in the brain. The methods developed will be applicable to the mouse, which is the leading mammalian genetic model system in health research. In addition to measuring chemical processes, the methods will also provide the ability to stimulate a specific population of neurons, which is important both in determining basic mechanisms of brain function and in evaluating new methods for neural prostheses.